Beef tallow injection matrix for serial crystallography

Ki Hyun Nam

doi:10.1038/s41598-021-04714-6

Abstract

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

Highlights

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage
Serial crystallography (SX) is a technique that enables the observation of room temperature structures of various molecules, ranging from macromolecules to small molecules[1,2,3,4]
Beef tallow can provide a relatively more stable injection stream in a temperature environment higher than room temperature compared to shortening and lard

Summary

Introduction

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. The liquid jet injection method has been widely used since the beginning of SX applications in research; this method is advantageous because it maintains a hydrated environment and a narrow diameter of the injection stream, resulting in very low background scattering[17] This sample delivery method requires a high flow rate to continuously and stably deliver the sample to the X-ray interaction point; in an X-ray free-electron laser facility with a low repetition rate or synchrotron, the number of unexposed crystals is overwhelmingly larger than that of crystals exposed to X-rays, leading to high sample consumption[16, 18, 23]. Sample delivery using viscous media maintains stable injection streams, even at low flow rates, because of the viscosity of the s ubstance[16] This method is widely applied in X-ray free-electron laser facilities with low repetition or synchrotron X-rays, as it can markedly reduce sample consumption compared to the liquid jet injection method. Aliphatic LCP (e.g. monoolein), which has been widely applied as a delivery medium for membrane proteins and water-soluble proteins, generates a stable stream even at very

Methods

Results

Conclusion